J Med Assoc Thai 2009; 92 (9):81

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Determination of the Apo E Genotype Using LightCyclerTM Apo E Mutation Detection Kit
Tirawanchai N Mail, Somkasetrin A , Avirutakarn C , Bunyaratvej N

Apolipoprotein E (Apo E) is found to be genetically polymorphic. There are 3 common alleles
designated as E2, E3 and E4. Polymorphism of the Apo E DNA is associated with the risk increase of many
diseases such as dyslipidemia, cardiovascular diseases, Alzheimer’s diseases, etc. Hence, the interest in
genotyping of the Apo E is now rising for the purpose of pre-diagnosis. The aim of this study was to characterize
the Apo E DNA polymorphism using LightCyclerApo E mutation detection kit (Roche) in the Thai
normal healthy subjects. The genomic DNA was extracted from the blood of 133 normal healthy subjects using
DNA extraction kit (Roche). Exon 4 of the Apo E gene was amplified by the extracted genomic DNA using the
real-time PCR. The simultaneous analysis of the two polymorphic codons (Codons 112 and 158) in a single
reaction was conducted by using the two reporter dyes with the different excitation and emission spectrum
LightCycler-Red 640 (LC-Red 640) and LC-Red 705 followed by the color compensation software to correct
the temperature-dependent crossover among the emission spectra of the dyes. The different genotypes were
then determined by performing the melting curve analysis in the two different channels. The results showed
that the allele frequencies of the Apo E2, E3 and E4 were 0.26, 0.63 and 0.11 respectively and the genotype
frequencies of the E2/3, E2/4, E3/3,E3/4 and E4/4 were 47.37, 5.26, 32.33, 14.28 and 0.75% respectively. This
study found that E2/3 was the most common genotype of the Apo E. In conclusion, the LightCycler (LC) allelic
discrimination method to genotype the Apo E is rapid, simple, reliable and strongly recommended for being
successful diagnostic tests in the future.

Keywords: Apolipoproteins E, DNA, Mutation, Polymorphism, Genetic, Reagent kits, Diagnostic

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